Various species of the protozoan family Trypanosomatidae are responsible for a range of serious human diseases in tropical and subtropical areas of the world. The subspecies Trypanosoma brucei is one of three known to cause sleeping sickness in sub-Saharan Africa, significantly contributing to the millions of people worldwide who are infected by these parasites and endangering hundreds of millions more. Many of the disorders caused by trypanosomatids are fatal if left untreated, but most currently used drugs are inefficient and toxic. ..more

Various species of the protozoan family Trypanosomatidae are responsible for a range of serious human diseases in tropical and subtropical areas of the world. The subspecies Trypanosoma brucei is one of three known to cause sleeping sickness in sub-Saharan Africa, significantly contributing to the millions of people worldwide who are infected by these parasites and endangering hundreds of millions more. Many of the disorders caused by trypanosomatids are fatal if left untreated, but most currently used drugs are inefficient and toxic.

Of many possible drug targets in trypanosomatid parasites, the carbohydrate metabolism pathway is seen as potentially one of the most selective, as T. brucei, when in the bloodstream of its mammalian host, is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply [1]. Oxidative metabolism involving the mitochondrial tricarboxylic acid cycle and oxidative phosphorylation are repressed in these parasites, and recent RNA interference (RNAi) experiments have shown that even partial depletion of certain individual glycolytic enzymes can lead to the death of cultured parasites [2]. One such glycolytic enzyme, phosphofructokinase (PFK), catalyzes the formation of fructose-1,6-bisphosphate (F1,6BP) and ADP from fructose-6-phosphate (F6P) and ATP, and in many metabolic circumstances makes an important contribution to the control of flux through the glycolytic pathway. As a specific glycolytic target, PFK is particularly attractive as it catalyzes the first irreversible step in glycolysis, and structural and kinetic studies have shown very substantial and essential differences from corresponding host enzymes [3], allowing for the discovery of parasite-selective inhibitors.

Substrate buffer was dispensed into white, solid 1536-well plates at 3ul/well in 0.1M triethanolamine (TEA) buffer, pH 8.0, containing final concentrations of 5 mM MgCl2, 0.01% Tween20, 0.5 mM fructose-6-phosphate (F6P) and 0.1mM ATP. Then, 23nl of compounds or DMSO were delivered to each well using a pin tool. One ul T. brucei PFK (0.1M TEA, 0.01% Tween20, 0.1% bovine serum albumin (BSA) and 1.25nM PFK, final concentrations) was then dispensed, and plates were incubated at room temperature for 45 min. The luminescent detection reagent, Kinase-Glo Plus (Promega), was then added at 2ul/well and incubated for ten minutes at room temperature. The plates were measured on a ViewLux plate reader for luminescent signal using a clear filter with a one second exposure. The %Activity was determined from the corrected luminescence values. As no specific T. brucei PFK inhibitors have been identified in the literature, 1x (1.25nM) and 0x PFK enzyme controls (untreated) were included to normalize %Activity of identified inhibitors; 0x enzyme values corresponded to 100%Activity (full inhibition), while 1x PFK enzyme values were used to normalize 0% activity (no inhibition). The Kinase-Glo Plus protocol varies from the primary screen in that it measures ATP, rather than ADP, concentration, and thus provides a measure of substrate depletion, rather than of product formation.

Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent increases in luminescence, concordant with a decrease in PFK activity and ATP depletion. Inversely, active activators showed a concentration-dependent decrease in luminescence signal below 1x PFK control levels, suggestive of an increase in ATP depletion. Inactive compounds showed no effect on luminescence signal.

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

6

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

11

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

12

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.